The coastal ocean is an important economic and recreational resource that is constantly influenced by human activities. In 2003, there were more than 18,000 days of beach closings throughout the US due to high concentrations of fecal bacteria. This was an increase of more than 51% over the previous year. Health related management of recreational coastal sites is currently undertaken by monitoring fecal coliform and enterococci by membrane filtration. Elevated concentrations of enterococci in marine waters have been shown to have a strong correlation with illness (especially gastrointestinal disease) in exposed individuals therefore making enterococci the indicator organism of choice for saline waters. The problem with this type of standard indicator monitoring is that there is a lag of at least 24-48 hours between when the sample is collected and when the data become available. Changes in the water quality and potential exposures during this delay lead to management decisions and public notifications that are often times inaccurate. To improve our management abilities, a primer pair and probe have been adapted for the large subunit ribosomal RNA gene of enterococci for use in a real-time nucleic acid sequence based amplification (NASBA) assay. This region is highly conserved within all reported species of the Enterococcus genus. Using this assay, the equivalent of less than one enterococci colony forming unit (CFU) was detected from a spiked sea water sample (100 ml). Further, there is a negative linear relationship (R2=0.9484) between CFUs obtained by membrane filtration and time to positivity (TTP) readings obtained with the NASBA assay. Therefore, quantitative estimates of enterococci are possible over at least four orders of magnitude and all positive samples amplified within forty-three minutes. By coupling this real-time NASBA assay to the inventors' existing field RNA extraction procedure and portable NASBA detection device, this technology will provide a simple, rapid (<1 hr), convenient testing format for coastal sites and greatly improve the health risk assessment of these regions.
Unlike polymerase chain reaction (PCR) based methods, NASBA is able to amplify RNA in a DNA background, and thus, only viable targets will be detected. This is important for indicator monitoring because only recent pollution events will be detected and false positive amplifications of naked DNA present in the sample will be eliminated. Indicator bacteria are chosen because of their ability to survive longer than the pathogenic organisms in question and therefore only viable bacteria should be considered as part of a risk analysis.